The Rockefeller University Press $30.00
J. Cell Biol. Vol. 192 No. 2 321–334
K.A. Myers and K.T. Applegate contributed equally to his paper.
Correspondence to Clare M. Waterman: firstname.lastname@example.org; Robert
S. Fischer: email@example.com; or Gaudenz Danuser: Gaudenz_Danuser@
Abbreviations used in this paper: ANOVA, analysis of variance; EC, endothelial
cell; HUVEC, human umbilical vein EC; MT, microtubule; PA, polyacrylamide;
ROI, region of interest.
Cell branching morphogenesis is critical to establishing a
functional vascular system. During angiogenesis, vascular endo
thelial “tip cells” lead a migrating chain of endothelial cells
(ECs). Tip ECs extend cell branches in response to directional
cues that guide their migration through the ECM to establish
the vascular network (Gerhardt et al., 2003, 2004; Gerhardt
and Betsholtz, 2005). Similar morphogenetic processes occur
during the establishment of the nervous system. In neurons,
cell branches (neurites) also extend from the cell body to form
axons and dendrites, both tipped by growth cones that are guided
by extracellular cues toward target cells to establish a functional
neuronal network (Dickson, 2002; Kalil and Dent, 2005). It is
well established that during neurite initiation, cell branching
is mediated by the coordinated remodeling of the actomyosin
and microtubule (MT) cytoskeletons (Dehmelt et al., 2003; Dent
and Gertler, 2003; Dehmelt and Halpain, 2004; Rösner et al.,
2007), but the mechanisms underlying EC branching morpho
genesis are less well understood. Myosin II contractility in the
cortical actin cytoskeleton is a negative regulator of neurite
initiation and elongation, as inhibition of myosin II or its up
stream activators promotes these processes, whereas myosin II
overexpression inhibits them (Kollins et al., 2009). Similarly, in
ECs, myosin II contractility negatively regulates branch initia
tion, as indicated by the formation of branches at sites of local
myosin II depletion in the cortex (Fischer et al., 2009). In neu
rons, MTs and their dynamic instability are required for neurite
initiation and extension, elaboration of the growth cone, and
be regulated by physical attributes of the extracellular
matrix (ECM) in a process termed mechanosensing.
Here, we tested the involvement of microtubules in linking
mechanosensing to endothelial cell branching morpho-
genesis. We used a recently developed microtubule plus
end–tracking program to show that specific parameters
of microtubule assembly dynamics, growth speed and
growth persistence, are globally and regionally modified
by, and contribute to, ECM mechanosensing. We demon-
strated that engagement of compliant two-dimensional or
uring angiogenesis, cytoskeletal dynamics that
mediate endothelial cell branching morphogen-
esis during vascular guidance are thought to
three-dimensional ECMs induces local differences in micro-
tubule growth speed that require myosin II contractility.
Finally, we found that microtubule growth persistence is
modulated by myosin II–mediated compliance mechano-
sensing when cells are cultured on two-dimensional ECMs,
whereas three-dimensional ECM engagement makes
microtubule growth persistence insensitive to changes
in ECM compliance. Thus, compliance and dimensionality
ECM mechanosensing pathways independently regulate
specific and distinct microtubule dynamics parameters in
endothelial cells to guide branching morphogenesis in
physically complex ECMs.
Distinct ECM mechanosensing pathways regulate
microtubule dynamics to control endothelial cell
Kenneth A. Myers,1 Kathryn T. Applegate,2 Gaudenz Danuser,2,3 Robert S. Fischer,1 and Clare M. Waterman1
1Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892
2Laboratory for Computational Cell Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
3Laboratory for Computational Cell Biology, Department of Cell Biology, Harvard Medical School, Boston, MA 02115
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T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 192 • NUMBER 2 • 2011 322
To better understand the mechanism of tip EC guidance
during angiogenesis, we sought to determine if MT dynamics
were regulated by ECM compliance and dimensionality, and
in turn, to explore the role of regionally regulated MT dynam
ics in mechanosensingmediated modulation of EC branch
ing morphogenesis. To accomplish this, we relied on a recently
developed computational image analysis method that tracks
the position of fluorescently tagged MT plus end–tracking pro
teins to derive independently regulated parameters of MT dy
namic instability, including growth rate and growth persistence
(1/catastrophe frequency). Our systematic measurements show
that in both 2D and 3D ECMs, compliance mechanosensing
regulates the MT growth rate through a myosin II–dependent
pathway. In contrast, compliance mechanosensing in 2D ECMs
regulates MT growth persistence, whereas in 3D ECMs, MT
growth persistence is insensitive to compliance. These results
indicate that distinct pathways regulate specific parameters of
MT dynamic instability via compliance and topology mechano
sensing to guide branching morphogenesis of ECs.
MT dynamics inhibit frequent cell branching
and promote rapid directional migration
We previously demonstrated that EC branching is enhanced
by softer (more compliant) ECMs (Fischer et al., 2009).
To determine if MT dynamics mediate complianceinduced
EC branching morphogenesis and migration, we investigated
the response of human umbilical vein ECs (HUVECs) to MT
perturbing drugs and 2D ECMs of different stiffness. HUVECs
were cultured on collagencoated glass or on either 8.7 kPa
(stiff) or 0.7 kPa (soft) collagencoupled polyacrylamide (PA)
substrates (Fig. 1 A). We analyzed branch frequency, defined
as the number of pseudopodial protrusions >10 µm in length
per unit cell area, as well as branch length. We first examined
the role of MTs in EC branching by incubating HUVECs with
20 µM nocodazole to inhibit MT assembly and promote dis
assembly. When cultured on glass or a stiff ECM, this treatment
increased branch frequency but had no effect on branch length.
Both branch length and branch frequency were reduced in
nocodazoletreated cells on soft ECMs (0.7 kPa), likely be
cause the cells spread poorly (Fig. 1, A and B). To determine the
role of MT disassembly in cell branching, we investigated the
effects of stabilizing MTs with 20 µM taxol. On both glass and
compliant ECMs, taxol treatment reduced branch length and
significantly increased branch frequency (P < 0.05; Fig. 1 B).
Because both nocodazole and taxol promoted frequent branch
ing in wellspread cells on stiff or soft ECMs, this suggests that
MT assembly/disassembly dynamics negatively regulate cell
branching, independent of ECM compliance.
We next sought to determine the role of MT assembly dy
namics in ECM compliancemediated modulation of cell mi
gration (Fig. 1 C). Unlike other studies (Lo et al., 2000; Fischer
et al., 2009; Petrie et al., 2009), we found that increasing ECM
compliance did not significantly influence HUVEC migration
velocity or directional persistence (defined as distance from
origin), an effect that is likely caused by the relatively high
axonal branching (Dent and Kalil, 2001; Dehmelt et al., 2003).
In contrast to the wealth of information on the role of MTs in
neuronal elaboration, the role of MTs in EC branching morpho
genesis is not well defined.
Although soluble and diffusible guidance cues have long
been known to regulate the cytoskeleton in cell morphogenesis
and migration during angiogenesis, regulation by the physical/
mechanical attributes of the ECM, a process termed “ECM
mechanosensing,” is now emerging as an important mechanism
(Ingber, 2002; Ghosh et al., 2008; Mammoto et al., 2009). Two
specific physical properties of the ECM, compliance (stiffness/
softness) and topology, can influence cell signaling and the
organization of the cytoskeleton to drive changes in cell mor
phology. In “compliance mechanosensing,” cells sense ECM
stiffness through cell–ECM focal adhesions, and respond by
modulating myosin II activity such that cell contractile forces
match the resistive compliance of the ECM (Pelham and Wang,
1997; Olson, 2004; Discher et al., 2005; Saez et al., 2005).
Accordingly, compliant (soft) ECMs promote downregulation
of myosin II activity and extension of cell branches in both ECs
and neurons, whereas stiff ECMs enhance myosin II activity to
limit cell branching (Flanagan et al., 2002; Fischer et al., 2009).
Cells were known to exhibit differential morphological
responses to 2D versus 3D ECM engagement (Beningo et al.,
2004; EvenRam and Yamada, 2005). This topologydependent
phenomenon is referred to as “ECM dimensionality mechano
sensing.” When cells engage a planar 2D ECM in tissue culture,
this defines their ventral surface and leaves their dorsal surface
unengaged. Here, many cells including ECs display a spread,
flattened morphology with actin stress fibers and peripheral
lamellipodia. In contrast, ECs embedded in a 3D ECM (Fischer
et al., 2009) or fibroblasts plated on planar 2D ECMs with local
dorsal ECM engagement (Beningo et al., 2004) display a spindle
shaped morphology, with long cell extensions tipped by tiny
lamellipodia (Cukierman et al., 2002; Doyle et al., 2009). Thus,
compliance and dimensionality mechanosensing induce major
changes in cell morphology that are likely mediated by specific
regulation of the cytoskeletal systems.
We showed recently that 3D ECM engagement by ECs syn
ergizes with ECM compliance to enhance cell branching, which
suggests that compliance and dimensionality mechanosensing
may mediate cell branching through distinct pathways (Fischer
et al., 2009). One possibility is that compliance and dimensionality
mechanosensing in ECs may affect cell branching morphogene
sis differentially through myosin II– or MTdependent pathways.
Indeed, evidence in other cell types suggests that MTs, like myo
sin II, may be regulated by ECM mechanosensing, and may also
mediate the morphological responses to mechanosensing (Kaverina
et al., 2002; Rhee et al., 2007). For example, MTs grow toward
sites of local ECM stiffening and retract in response to locally
applied contraction inhibitors (Kaverina et al., 2002). In compli
ant 3D ECMs, MTs are required for branching morphology of
fibroblasts, whereas in stiff 3D ECMs, MTs are not required for
branching but are needed for cell polarization (Rhee and Grinnell,
2007). However, whether MT assembly dynamics are regulated
by independent compliance or topology mechanosensing path
ways to mediate EC branching morphogenesis is not known.
323ECM mechanosensing regulates MT dynamics and EC branching • Myers et al.
(Fig. 2, A and C). In addition, MT dynamics data from indi
vidual whole cells or userdefined subcellular regions can be
pooled to allow quantitative measurements of variations in
MT track density (Fig. S1 A), as well as statistical analysis of
changes in MT dynamics behavior under different experimental
conditions or in different subcellular regions that may not be
readily apparent by eye (Video 4 and Fig. S1, B and C). Total
MT track numbers were correlated with the mean cell area for
individual experimental conditions (Fig. S2), and thresholds for
classifying EB3 tracks as “slow” versus “fast” or “shortlived”
versus “longlived” were based on the mean value for each param
eter from the entire population of cells analyzed over all experi
mental conditions (Fig. 2 B and Table S1).
To test the hypothesis that ECM compliance mechano
sensing regulates MT assembly dynamics, we analyzed GFPEB3
dynamics in HUVECs cultured on substrates of varying compli
ance. To maximize the sensitivity of these experiments, we com
pared MT dynamics in cells cultured on collagencoated glass with
cells cultured on soft (0.7 kPa) collagencoated PA substrates
(Table I and Table S1). In cells adhered to glass, MT growth excur
sions were primarily slow (Fig. 2 D, red + green), and the majority
of slow excursions were also shortlived (red), whereas on more
compliant ECMs, a greater proportion of MTs had fast growth
excursions (Fig. 2 D, yellow + blue). Qualitative examination of
image overlays of colorcoded MT plus end motion tracks for
individual cells suggested that fast, longlived growth may be
concentrated in the cell center (Fig. 2 C, blue tracks), and that fast,
variability in directional movement and instantaneous veloci
ties of HUVECs compared with other cell types. Nevertheless,
treatment of cells on either stiff or soft ECMs with either no
codazole or taxol significantly reduced both migration velocity
and directional persistence (Fig. 1 C and Videos 1–3). Together,
these data suggest that MT assembly/disassembly dynamics re
strict excessive cell branching to promote fast, directional cell
migration independent of ECM compliance.
Increased ECM compliance promotes fast-
growing, dynamically unstable MTs
Because both MT assembly/disassembly dynamics and ECM
compliance (Fischer et al., 2009) influence cell branching, we
reasoned that ECM compliance mechanosensing could regu
late MT assembly dynamics to modulate cell branching. To test
the effects of substrate compliance on MT dynamics, we per
formed highresolution livecell imaging of fluorescently la
beled EB3, which tracks with the growing plus ends of MTs.
We used our recently developed software package plusTip
Tracker (Matov et al., 2010), which enables highthroughput
measurement of MT growth speed and growth persistence
(1/catastrophe frequency), in timelapse image series of fluor
escent MT plus end–binding proteins (Fig. 2 A). Data output
from plusTipTracker includes image overlays of MT plus end
motion tracks, with the magnitudes of MT growth speed and
growth lifetime colorcoded to allow qualitative visualiza
tion of regional differences in these values throughout the cell
Figure 1. Perturbation of MT growth or shortening promotes HUVEC branching and inhibits rapid directional migration. (A) Immunolocalization of MTs
and fluorescent phalloidin staining of actin in HUVECs cultured on collagen-coated glass, or stiff (8.7 kPa) or soft (0.7 kPa) compliant ECMs. Samples were
treated with DMSO (control), 20 µM nocodazole (Noc.), or 20 µM taxol for 90 min. Bars, 20 µm. (B) Analysis of the effects of the treatments in A on cell
branch frequency and length. (C) Analysis of the effects of the treatments in A on cell migration velocity and distance to origin. *, P < 0.05 comparing
compliance versus compliance + drug. **, P < 0.05 compared with glass (one-way ANOVA). Error bars indicate standard deviation.
JCB • VOLUME 192 • NUMBER 2 • 2011 324
Fischer et al., 2009). Therefore, we sought to determine if
ECM complianceinduced effects on MT dynamics were caused
by compliance mechanosensingmediated downregulation of
myosin II activity. In HUVECs cultured on glass and treated
with 20 µM blebbistatin to inhibit myosin II ATPase activity
(Fig. 2 D), MTs displayed an increase in the proportion of both
fast, shortlived (yellow) and fast, longlived (blue) growth
excursions, and a reduction in the proportions of both slow,
shortlived (red) and slow, longlived (green) growth, resulting
in an overall trend that was similar to the effects of compli
ant ECMs on MT dynamics (Fig. 2 D, compare 0.7 kPa and
Glass + Blebb). Blebbistatin treatment and ECM compliance
also similarly increased mean MT growth speed and decreased
mean growth lifetime compared with cells on glass (P < 0.001;
shortlived growth may be concentrated in the cell periphery
(Fig. 2 C, yellow tracks). These results were confirmed via analy
sis of MT dynamics in specific subcellular regions (Fig. 3 and
Fig. S1). Together, the effects of increased compliance in ECs cul
tured on 2D ECMs resulted in a significantly greater mean MT
growth speed and a significantly lower mean MT growth lifetime
(P < 0.001; Fig. 2 E and Table I). Thus, ECM compliance mechano
sensing reduces MT growth persistence (i.e., promotes dynamic
instability) and promotes fast MT assembly.
Effects of ECM compliance on MTs are
myosin II dependent
It is well established that cell engagement of compliant ECMs
downregulates myosin II contractility (Discher et al., 2005;
Figure 2. Down-regulation of myosin II by compliance mechanosensing promotes fast, short-lived MT growth excursions. (A) Workflow used in the
plusTipTracker software package for detecting fluorescent EB3 comets, tracking their movement, and classifying MT growth dynamics based on growth
speed and growth lifetime. (B) Color scheme for the four subpopulations of MT growth tracks derived by plusTipTracker software and depicted in C and D.
(C) Color-coded MT growth track subpopulation overlays from 2 min time-lapse movies of GFP-EB3 (frame rate = 2 s) on representative cells for comparison
between a cell plated on glass with (Glass + Blebb) or without (Glass) 20 µM blebbistatin treatment, or on a more compliant ECM (0.7 kPa). Bars, 10 µm.
(D) Percentage of the population of MTs whose dynamics were categorized in the four subpopulations described in B in cells under the conditions described
in C. (E) Comparison of percentages of mean MT growth speeds and growth excursion lifetimes in cells under the conditions described in C. *, P < 0.001.
Error bars indicate standard error of the mean.
325ECM mechanosensing regulates MT dynamics and EC branching • Myers et al.
(31% increase) and in the cell body (43% increase; Fig. 3 C).
This resulted in increased mean growth speeds in both branches
and the cell body compared with the same regions of the cells
on glass (*, P < 0.001; Fig. 3 D, left; and Table I). In contrast,
ECM compliance tended to increase MT growth lifetime in
branches and significantly decrease growth lifetime in the cell
body compared with the same regions of cells on glass. As a
result, growth lifetimes that were regionally distinct on glass
substrates (**, P < 0.001; Fig. 3 D, right) became similar in
branches and the cell body on compliant substrates (Fig. 3 D,
right; and Table I). These results suggest that compliance mechano
sensing promotes fast MT assembly globally, and reduces growth
lifetimes specifically in the cell body.
To determine if complianceinduced regional effects
on MT assembly dynamics were caused by downregulation
of myosin II activity (Fischer et al., 2009), we compared re
gional MT dynamics in blebbistatintreated cells plated on
glass to those in cells on 0.7 kPa ECMs. This revealed that
ECM compliance and myosin II inhibition produced simi
lar effects on MT dynamics in both the cell body and cell
branches (P = 0.022; Fig. 3 D), enhancing growth speed glob
ally and specifically reducing growth lifetime in the cell body
compared with the same regions of cells on glass. Together,
these data suggest that downregulation of myosin II by ECM
compliance mechanosensing promotes fast MT growth glob
ally throughout the cell, but homogenizes regional differences
in MT growth lifetimes by locally increasing MT dynamic
instability in the cell body and locally increasing persistent
MT growth in cell branches.
MT dynamic instability regulates cell
branching and migration similarly
in 2D and 3D
Our previous studies in primary mouse ECs showed that the
combined effects of increased substrate compliance and 3D
ECM engagement synergistically promote cell branching,
which suggests that compliance and 3D ECM engagement
promote branching via distinct pathways (Fischer et al., 2009).
We hypothesized that 3Dspecific effects on cell branching
may be mediated through the MT cytoskeleton. To control the
compliance of the in vitro 3D environment, we covalently
coupled 3D collagen matrices to PA gels of defined shear
modulus and affixed these to microscope coverslips to make
collagen/PA/glass “sandwich cultures” (Fischer et al., 2009).
ECs at the PA–collagen interface engage collagen on their
ventral surface, which is covalently coupled to PA of a defined
stiffness, but also engage dorsal collagen in the 3D matrix.
Although the cells experience anisotropic stiffness (the colla
gen matrices are softer than the underlying PA), their behav
ior is dominated by the maximum stiffness encountered (Lo
et al., 2000; Guo et al., 2006), which is that of the collagen
coupled to the PA.
To determine if MT assembly/disassembly dynamics are
important for the effects of compliance and 3D ECM engage
ment on cell branching morphogenesis and migration, we ana
lyzed HUVEC branching in 3D sandwich cultures of variable
compliance (Fig. 4 A). We first confirmed that, like primary
Fig. 2 E and Table I). Together, our results suggest that down
regulation of myosin II by ECM compliance mechanosensing
promotes fastgrowing, dynamically unstable MTs.
Regional modulation of myosin II by
compliance mechanosensing promotes
fast MT growth globally, but differentially
regulates MT growth persistence in cell
branches and the cell body
Qualitative examination of the spatial distribution of MT growth
tracks (Fig. 2 C, colored tracks) suggested that cells may re
spond to increased ECM compliance by differentially modulat
ing MT assembly dynamics in subcellular regions (Wittmann
and WatermanStorer, 2005). To test this hypothesis, we com
pared measurements of MT dynamics in cell branches (Fig. 3 A,
orange regions) to measurements of MT dynamics in the pe
ripheral cell body (Fig. 3 A, purple region). Because MTs grow
ing from the centrosome to the peripheral cell body are known
to have dynamics different from those in membraneproximal
regions (Komarova et al., 2002; Wittmann and WatermanStorer,
2005), we excluded data from the cell center to focus our com
parison on local regulation of membraneproximal cell regions.
We first analyzed regional MT regulation in cells on ECM
coated glass where branches are few and short. This revealed
that the majority of MT growth excursions within branches
were slow and shortlived, whereas in the peripheral cell body,
MT growth excursions were similarly slow, but with signifi
cantly longer growth lifetimes (*, P < 0.001; Fig. 3 B). Thus, on
glass, MT dynamics are regionally regulated such that more
persistent growth occurs in the peripheral cell body, whereas in
cell branches, MTs tend to be dynamically unstable.
Comparison of regional MT dynamics in cells on compli
ant (0.7 kPa) ECMs to those in cells on glass revealed an in
crease in the proportion of fast MT growth excursions, both
short and longlived (yellow and blue), in both cell branches
Table I. MT growth dynamics in 2D ECMs
ECM condition n Mean speed Mean lifetime
2D 0.7 kPa
Glass + Blebb
2D 0.7 kPa
Glass + Blebb
2D 0.7 kPa
Glass + Blebb
5.02 ± 0.254
8.90 ± 0.282
9.10 ± 0.323
9.65 ± 0.596
8.71 ± 0.366
7.66 ± 0.354
18 5.10 ± 1.08
8.52 ± 0.344
8.62 ± 0.299
9.56 ± 1.17
11.6 ± 0.488
9.82 ± 0.394
4.93 ± 0.237
9.55 ± 0.425
9.39 ± 0.535
13.3 ± 0.747
11.8 ± 0.667
10.5 ± 0.744
MT growth speeds and MT growth lifetimes in HUVECs cultured on 2D ECMs
calculated for the entire cell area (whole cell) and subcellular regions (cell
branches [Branches] vs. peripheral cell body [Periphery]). ECM condition re-
fers to collagen-coated glass with (Glass + Blebb) or without (Glass) treatment
with 20 µM blebbistatin or collagen coupled to PA of 0.7 kPa shear modulus
(0.7 kPa). Mean values reported are ± standard error of the mean. n, number
of MT growth tracks.
JCB • VOLUME 192 • NUMBER 2 • 2011 326
Compliance mechanosensing promotes fast
MT growth in both 2D and 3D ECMs
To test the hypothesis that 3D ECM engagement modulates MT
dynamics, we compared MT dynamics in cells on 2D ECMs with
cells in 3D sandwich cultures of the same PA stiffness (8.7 kPa;
Fig. 5 A). Compared with 2D ECMs, in 3D sandwich cultures a
greater proportion of MTs had fast, shortlived growth excursions
(Fig. 5 B and Fig. S3), which appeared concentrated near the cell
periphery (3D 8.7 kPa; Fig. 5 A). This resulted in a significantly
greater mean MT growth speed and a significantly lower mean
MT growth lifetime in cells in 3D compared with 2D ECMs
(P < 0.001; Fig. 5 D and Table II). These data suggest that com
pared with 2D ECMs of the same stiffness, 3D ECM engagement
promotes fast and dynamically unstable MT growth.
mouse ECs (Fischer et al., 2009), engagement of compliant
(0.7 kPa) 3D ECMs in sandwich cultures strongly enhanced
HUVEC branching frequency compared with stiffer (8.7 kPa)
2D ECMs and 3D sandwich cultures (P < 0.05; Fig. 4 B). Phar
macological perturbations revealed that, similar to results in 2D
ECMs (Fig. 1, B and C), treatment of cells in 3D sandwich cul
tures of different stiffnesses with either taxol or nocodazole in
creased cell branching frequency but had little effect on branch
length (Fig. 4 B), and reduced both migration velocity and di
rectional persistence (Fig. 4 C and Videos 5 and 6). Together
these data suggest that MT assembly/disassembly dynamics
suppress excessive EC branching in both 2D and 3D ECMs,
and promote fast, directional migration, independent of compli
ance and dimensionality.
Figure 3. Down-regulation of myosin II by compliance mechanosensing promotes fast MT growth globally and short-lived growth excursions in the periph-
eral cell body. (A) Workflow for categorizing GFP-EB3 comet tracks of MT growth into specific subcellular regions including cell branches and the peripheral
cell body. A color code key for the classification of MT growth excursion subpopulations is shown below. Bar, 10 µm. (B) Comparison of percentages (top)
of the population of MTs whose growth dynamics were categorized in the four subpopulations described in A, and MT growth speeds and growth excursion
lifetimes (bottom) in branch and peripheral cell body regions of cells plated on glass coverslips. (C) Comparison of percentages of the population of MTs
whose growth dynamics were categorized in the four subpopulations described in A in branch and peripheral cell body regions. Cells were plated on glass
with or without the addition of 20 µM blebbistatin (Glass + Blebb) or on compliant ECMs (0.7 kPa). Percentage values are shown below. (D) Comparison
of MT growth speeds (left) and growth excursion lifetimes (right) in branch and peripheral cell body regions of cells plated on glass with or without the
addition of 20 µM blebbistatin (Glass + Blebb) or on compliant ECMs (0.7 kPa). **, branches versus cell body; *, between group comparison, P < 0.001.
Error bars indicate standard error of the mean.
327ECM mechanosensing regulates MT dynamics and EC branching • Myers et al.
that downregulation of myosin II by compliance mechanosens
ing promotes fast MT growth in both 2D and 3D ECMs.
3D ECM engagement uncouples compliance
mechanosensing from myosin II–mediated
regulation of MT growth persistence
Because we observed in 2D cultures that compliance signifi
cantly reduced MT growth lifetimes (Fig. 2 E and Table S1, com
pare glass vs. 0.7 kPa), we also compared MT growth lifetimes
in 3D sandwich cultures of three different stiffnesses. Interest
ingly, unlike in 2D, when comparing cells in compliant 3D
ECMs to cells in stiffer 3D ECMs, the proportion of MTs
exhibiting shortlived or longlived MT growth excursions were
similar (Fig. 5 C and Fig. S3), and the mean MT growth lifetimes
were not significantly different (Fig. 5 E and Table II). Thus, 3D
ECM engagement makes MT growth persistence insensitive to
compliance. However, despite the insensitivity of MT growth
persistence to compliance in 3D sandwich cultures, we found
that in 3D ECMs, growth persistence was still sensitive to direct
inhibition of myosin II via blebbistatin. As shown in Fig. 5 C,
myosin II inhibition in 3D promoted an increase in the propor
tion of fast, shortlived MT growth excursions, and reduced the
proportion of both longlived and shortlived slow MTs, similar
To determine if compliance mechanosensing modulates
MT dynamics in cells in 3D ECM, we compared MT dynamics
in cells in 3D sandwich cultures of three different stiffnesses
(55 kPa, 8.7 kPa, and 0.7 kPa). For 3D studies, we used very stiff
(55 kPa) PA substrates instead of glass to evaluate the effects of
3D ECM engagement because it is necessary to conjugate the
collagen sandwich gel to a PA substratum. Examining the propor
tions of MTs in different dynamics classes revealed that, similar
to effects in 2D (Fig. 2 D), increasing compliance in 3D increased
the proportion of MTs exhibiting fast growth (Fig. 5 B), which
resulted in a significant increase (P < 0.001) in mean MT growth
speed compared with MTs in cells in stiffer 3D ECMs (Fig. 5 D).
Given that changes in compliance in 3D sandwich cultures alter
myosin II activity (Fischer et al., 2009), it seemed likely that myo
sin II activity may contribute to the increase in MT growth speed
observed in compliant 3D ECMs. To confirm this, we compared
MT dynamics in cells in compliant 3D sandwich cultures with
and without blebbistatin. This revealed that, as in 2D (Fig. 2 D),
myosin II inhibition in 3D ECMs increased the proportion of fast,
shortlived MT growth excursions and reduced the proportion
of both longlived and shortlived slow MT growth excursions
(Fig. 5 C and Fig. S3). This resulted in a significantly greater mean
MT growth speed (Fig. 5 E and Table II). These results suggest
Figure 4. Perturbation of MT growth or shortening affects cell branching and migration similarly in 2D and 3D ECMs. (A) Immunolocalization of MTs and
fluorescent phalloidin staining of actin in HUVECs cultured in 8.7 kPa or 0.7 kPa compliant 3D ECMs and treated for 90 min with DMSO vehicle (control),
20 µM nocodazole (Noc.), or 20 µM taxol. Bars, 20 µm. (B) Analysis of the effects of the treatments in A on cell branch frequency and length compared
with similar drug treatments of cells plated on 2D 8.7 kPa ECMs. (C) Analysis of the effects of the treatments in A on cell migration velocity and distance
to origin compared with similar drug treatments of cells cultured on 2D 8.7 kPa ECMs. *, P < 0.05 comparing compliance versus compliance + drug.
**, P < 0.05 compared with 2D 8.7 kPa ECMs (one-way ANOVA). Error bars indicate standard deviation.
JCB • VOLUME 192 • NUMBER 2 • 2011 328
effects throughout the cell and caused mean MT growth life
time to decrease (P < 0.001; Fig. 5 E and Table II). Thus, global
inhibition of myosin II activity promotes global increases in
MT growth speed and dynamic instability in both 2D ECMs
and 3D sandwich cultures. These results suggest that 3D ECM
engagement uncouples compliance mechanosensing from myo
sin II–mediated regulation of MT growth persistence. Together,
our results suggest that ECM compliance and dimensionality
mechanosensing regulate distinct, specific parameters of MT
dynamic instability; MT growth speed (i.e., assembly rate) is
regulated by ECM compliance independent of dimensionality,
but MT growth persistence (i.e., catastrophe frequency) is differ
entially regulated by ECM compliance and dimensionality.
MT growth speed, but not growth
persistence, is regionally regulated by
compliance mechanosensing in 3D ECMs
To determine if 3D ECM engagement modulates MT dynamics
within distinct subcellular compartments, we compared regional
MT dynamics between cells on 2D ECMs and 3D sandwich cul
tures of the same stiffness. First, we documented that like in 2D,
in 3D, MTs in cell branches grew more slowly and persistently
than MTs in the peripheral cell body (Fig. 6 A). Comparison of
regional MT dynamics between cells on 2D ECMs or 3D sand
wich cultures of the same stiffness (8.7 kPa) revealed that 3D
ECM engagement promoted a significant increase in mean MT
growth speed only in the cell body (P < 0.001), whereas growth
speeds in branches and growth lifetimes in both cell regions were
similar in cells in 2D ECMs and 3D sandwich cultures (Fig. 6 B
and Table II). Thus, 3D ECM engagement promotes fast MT
growth specifically in the cell body and not in cell branches.
We next sought to determine if compliance mechano
sensing or myosin II activity regionally modulates MT assembly
dynamics in cells in 3D ECMs. To first determine the effects of
compliance mechanosensing, we compared regional MT dynam
ics in cells in 3D sandwich cultures of three different stiffnesses.
This showed that increased compliance in 3D enhanced the pro
portion of fastgrowing MTs in both cell branches and the periph
eral cell body (Fig. 6 C). In contrast to 2D ECMs (Fig. 3), on the
most compliant (0.7 kPa) 3D ECMs, mean growth speeds were
significantly increased specifically within the peripheral cell body,
but not in cell branches. This demonstrates a 3D ECMspecific
effect on regional regulation of MT growth speed (**, P < 0.001;
Fig. 6 D, left; and Table II). Furthermore, regional differences
in MT growth speeds were only observed in 3D sandwich cul
tures of intermediate or greater compliance (8.7 kPa or 0.7 kPa;
**, P < 0.001). In the stiffest (least compliant) 3D sandwich cul
tures (55 kPa), where myosin II activity is high, or when myosin II
was inhibited directly with blebbistatin, no regional differences in
MT growth speed were observed. This suggests that regional reg
ulation of MT growth speed functions in a limited dynamic range
of myosin II activity in 3D. Similar to analysis of MT dynam
ics in whole cells, compliance of 3D sandwich cultures had no
effect on MT growth lifetimes in either cell body or branch re
gions (Fig. 6 D, right; and Table S1). Thus, compliance mechano
sensing does not regionally regulate MT growth persistence in 3D
ECM, but has regionspecific effects on MT growth speed.
to the effects of blebbistatin in 2D cultures (Fig. 2 D and Fig. S3,
compare glass vs. glass + Blebb). Also similar to cells in 2D,
blebbistatin treatment of cells in 3D ECMs produced these
Figure 5. Compliance mechanosensing promotes fast MT assembly in
both 2D and 3D, but 3D ECM engagement makes MT growth persistence
insensitive to compliance. (A) Color-coded MT growth track subpopulation
overlays from 2-min time-lapse movies of GFP-EB3 (frame rate = 2s) on
representative cells cultured on 8.7 kPa or 0.7 kPa 2D ECMs, for com-
parison with 8.7 kPa or 0.7 kPa 3D ECMs or with cells cultured in 8.7 kPa
3D ECMs and treated with 20 µM blebbistatin (3D 8.7 kPa + Blebb).
A color code key for MT growth dynamics classifications is shown below.
Bars, 10 µm. (B and C) Comparison of percentages of the population
of MTs whose growth dynamics were categorized in the four subpopula-
tions described in A in cells under the conditions described in A or on
55 kPa 3D ECMs. Percentage values shown below. (D and E) Comparison
of mean MT growth speeds and growth excursion lifetimes in cells under
the conditions described in A or on 55 kPa 3D ECMs. *, P < 0.001. Error
bars indicate standard error of the mean.
ECM mechanosensing regulates MT dynamics and EC branching • Myers et al.
where a cell branch was initiating (Fig. 7 B, left arrow) and
slower, longerlived growth (green tracks) in a branch that later
retracted before continuing to elongate (Fig. 7 B, arrowhead).
During elongation of existing branches, qualitative examination
of MT tracks suggested that MTs grew more slowly and persis
tently toward elongating branch tips (Fig. 7 B, arrows) than at
the base of the same branch. Together, these results suggest that
local modulation of MT growth speed in the cell body may lo
cally induce branch formation, whereas slower, more persistent
MT growth in cell branches may promote branch elongation.
Mechanosensing of the ECM is gaining importance as a poten
tial physiological regulator of EC branching morphogenesis
and motility during formation of vasculature (Ingber, 2002;
Ghosh et al., 2008). We recently showed that 3D ECM dimen
sionality mechanosensing by ECs synergizes with ECM com
pliance mechanosensing to enhance cell branching, which
suggests that compliance and dimensionality regulate distinct
molecular pathways of cell branch formation (Fischer et al.,
2009). Here, we tested the hypothesis that compliance and
dimensionality mechanosensing in ECs may affect cell branch
ing morphogenesis differentially through myosin II– and MT
dependent pathways. This work produced three main advances.
First, we used a recently developed MT plus end–tracking pro
gram to show that specific parameters of MT assembly dynam
ics, growth speed and growth persistence, are globally and
regionally modified by and contribute to ECM compliance and
dimensionality mechanosensing. Second, we demonstrated that
engagement of compliant 2D or 3D ECMs induces local differ
ences in MT growth speed that require myosin II contractility.
Finally, we showed that MT growth persistence is modulated by
myosin II–mediated compliance mechanosensing when ECs are
cultured on 2D ECMs, whereas 3D ECM engagement makes
MT growth persistence insensitive to changes in ECM compli
ance. Thus, compliance and dimensionality ECM mechano
sensing pathways independently regulate specific and distinct
MT dynamics parameters in ECs to guide branching morpho
genesis in physically complex ECMs.
Our results reveal new relationships between ECM com
pliance, topology mechanosensing, and MT dynamic instabil
ity, which are summarized in Fig. 7 C. On the stiffest 2D ECMs
(glass), ECs extend few branches, myosin II–mediated cortical
tension is uniformly high (Fischer et al., 2009), MT growth is
uniformly slow, and MT growth persistence is highest within the
cell body. On more compliant 2D ECMs (Fig. 7 C, 2D ECM,
0.7 kPa), reduction of myosin II contractility and spatial inhomo
geneity in cortical tension promotes branch initiation (Fischer
et al., 2009). This reduction in myosin II contractility also pro
motes faster, less persistent MT growth in the peripheral cell
body, which likely further modulates cortical contractility to en
hance cell branching. Additional inhibition of myosin II contrac
tility with blebbistatin (Fig. 7 C, 2D ECM, Glass + Blebb) further
promotes branch frequency while at the same time reducing MT
growth persistence. This revealed that both cell branching mor
phology and MT dynamics are responsive to a range of myosin II
MT growth speed is directly correlated
with branch frequency and inversely
correlated with branch elongation
To determine if specific parameters of MT assembly/dis
assembly dynamics regulate cell branching morphogenesis, we
examined the relationships between parameters of MT dy
namics and cell branching across all experimental conditions.
This revealed significant correlations between MT growth
speed and branch frequency, with a direct correlation between
MT growth speed and cell branching (ratio = 2.5:1; r = 0.893;
P < 0.01), and an inverse correlation between MT growth
speed and branch length (ratio = 19.5:1; r = 0.892;
P < 0.01; Fig. 7 A). The correlation coefficients were even
higher for data specifically from cell branches (Fig. 7 A, red
trend lines). In contrast, we found no significant correlations
between MT growth persistence and branch frequency or branch
length (Fig. S4). These results show that fast MT growth cor
relates with frequent cell branching, whereas slow MT growth
correlates with branch elongation.
To determine if local modulation of MT dynamics medi
ates cell branching in real time, we analyzed MT dynamics
during cell branch formation and/or elongation by timelapse
imaging of EB3GFP in cells plated on compliant 3D ECMs to
promote frequent branching. Because branch formation occurs
infrequently (branch initiations at 10min intervals) relative to
the time scales of MT dynamic instability (growth excursion ini
tiations at 10s intervals) and GFP photobleaching (minutes),
it was rare to capture highquality movies suitable for accurate
tracking of MT dynamics during new branch formation (Video 8).
However, analysis of EB3GFP tracks in the example shown in
Fig. 7 B revealed that MTs displayed locally faster and shorter
lived growth (yellow tracks) at a site in the peripheral cell body
Table II. MT growth dynamics in 2D versus 3D ECMs
ECM Condition n Mean speedMean lifetime
3D 8.7kPa + Blebb
3D 8.7kPa + Blebb
3D 8.7kPa + Blebb
8.82 ± 0.222
10.7 ± 0.409
11.7 ± 0.463
14.0 ± 0.480
10.4 ± 0.394
9.79 ± 0.476
9.67 ± 0.460
7.80 ± 0.387
8.42 ± 0.200
9.30 ± 0.207
11.2 ± 0.220
15.2 ± 0.233
14.7 ± 0.551
13.7 ± 0.353
13.4 ± 0.270
11.7 ± 0.267
9.14 ± 0.296
11.5 ± 0.523
13.5 ± 0.613
14.5 ± 0.519
13.2 ± 0.629
12.7 ± 0.673
12.5 ± 0.678
10.5 ± 0.506
MT growth speeds and MT growth lifetimes in HUVECs cultured on 2D ECMs
or in 3D collagen-PA-glass sandwich cultures calculated for the entire cell area
(whole cell) and subcellular regions (cell branches [Branches] vs. peripheral cell
body [Periphery]). ECM condition refers to collagen coupled to PA of 8.7 kPa
shear modulus (2D 8.7 kPa) or a 3D collagen gel coupled to PA of a defined
shear modulus with (3D 8.7 kPa + Blebb) or without treatment with 20 µM bleb-
bistatin (3D 8.7 kPa or 3D 0.7 kPa). Mean values reported are ± standard error
of the mean. n, number of MT growth tracks.
JCB • VOLUME 192 • NUMBER 2 • 2011 330
contractility. MT dynamics are also required for cell branching
morphology in response to compliance mechanosensing, which
suggests a potential feedback mechanism between myosin II
contractility and the regulation of MT dynamics in this process.
When cells engage stiff 3D ECMs, myosin II–mediated
compliance mechanosensing is additionally enhanced (Beningo
et al., 2004; Fischer et al., 2009), further augmenting MT growth
speeds compared with 2D ECMs (Fig. 7 C). As compliance is
increased in 3D ECMs, both cell branching and MT growth
speeds further increase. Indeed, over a range of experimental
conditions in 2D and 3D ECMs, MT growth speeds and cell
branching are strongly correlated (Fig. 7 A). However, when
cells engage 3D ECMs, MT growth persistence no longer re
sponds to changes in substrate compliance, but still responds to
direct myosin II inhibition. This suggests that 3D ECM engage
ment uncouples compliance mechanosensing from myosin II–
mediated regulation of MT growth persistence. One possible
interpretation of this is that 3D ECM engagement may specifi
cally regulate catastrophe factors, such as MCAK or Op18/
stathmin (van der Vaart et al., 2009), to regulate MT dynamics.
Although it is known that integrin engagement can regulate MT
dynamics (Palazzo et al., 2004), our results show that different
topologies of integrin engagement can differentially regulate
Our findings also demonstrate that unlike MT growth persis
tence, MT growth speeds are regionally regulated in branches and
bodies of cells on compliant substrates with 3D ECM engagement.
This suggests that the regional regulation of MT assembly/
disassembly factors, such as XMAP215 or Op18/stathmin
(van der Vaart et al., 2009), may be downstream of myosin II
contractility. Regional regulation of MT growth speed was lost
in either very stiff (55 kPa) 3D sandwich cultures, or when
myosin II activity was directly inhibited by blebbistatin. Because
we have previously shown that myosin II is more dynamic in
the cortex of cells in compliant 3D ECMs (Fischer et al., 2009),
this suggests that local differences in MT growth speed are likely
caused by local stochastic fluctuations of myosin II activity at
the cell cortex. We hypothesize that these stochastic fluctuations
of myosin II at the cell cortex may alter the local activity of
MT assembly factors, although the mechanism for this remains
unclear. We suggest that local differences in myosin II contrac
tility promote local differences in MT dynamics, which in turn
feed back to further modulate actomyosin through Rho and Rac
signaling (Rodriguez et al., 2003). This feedback may initiate
the spatially confined breaking of cortical tension that permits
branch formation during EC branching morphogenesis.
Materials and methods
Cells and DNA expression constructs
HUVECs were maintained in endothelial cell basal medium (EBM) supple-
mented with EGM-MV Single Quots (Lonza) at 37°C in 5% CO2. For live
imaging, medium was supplemented with 25 µg Hepes, pH 7.2, and
Figure 6. Compliance mechanosensing does not regionally regulate
MT growth lifetimes in 3D ECMs. (A) Comparison of percentages of the
population of MTs whose growth dynamics are categorized in four sub-
populations (key and percentage values are shown below) in cell branch
(left) and peripheral cell body (right) regions. (B) Comparison of mean
MT growth speeds (left) and growth excursion lifetimes (right) of cells
cultured on 2D or in 3D sandwich gels of the same stiffness (8.7 kPa).
(C) Comparison of percentages of the population of MTs whose growth
dynamics were categorized in the four subpopulations described in A
in cell branch (left) and peripheral cell body (right) regions (percentage
values are shown below). (D) A comparison of mean MT growth speeds
(left) and growth excursion lifetimes (right) of cells cultured in 8.7 kPa
3D sandwich gels with (3D 8.7 kPa + Blebb) or without the addition of
20 µM blebbistatin (3D 8.7 kPa) or in more compliant 3D sandwich gels
(3D 0.7 kPa). **, branches versus cell body; *, between group compari-
son, P < 0.001. Error bars indicate standard error of the mean.
331ECM mechanosensing regulates MT dynamics and EC branching • Myers et al.
in 0.5% 3-aminopropyltrimethyoxysilane diluted in water (10 min on a
stir plate), followed by 6 × 5 min water washes. Coverslips were dried
at 37°C, cooled to room temperature, and then immersed in 0.5% glutar-
aldehyde solution in PBS on a stir plate for 30 min. Coverslips were then
washed three times for 10 min in water and dried at room temperature.
For the experiments in this manuscript, PA gels of varying stiffness (55 kPa,
8.7 kPa, or 0.7 kPa; shear stress modulus) were prepared by varying
concentrations of 40% acrylamide and 2% bis-acrylamide before tetra-
methylethylenediamine polymerization (Gardel et al., 2008).
To generate 3D ECMs of controlled compliance (sandwich gels;
Fischer et al., 2009), coverslips with adhered PA gels were activated with
2 mM sulfo-SANPAH by exposure to 7,500 J of UV light, rinsed, and cova-
lently cross-linked to a thin layer of unpolymerized rat tail type I collagen
30 U/ml Oxyrase. Transfection of GFP- or mApple-EB3 cDNAs (courtesy of
M. Davidson, Florida State University, Tallahassee, FL) was performed using
a nucleofector (Amaxa Biosystems) with solution kit IV (Lonza), setting A-034,
and experiments were performed 6–10 h later to allow time for EB3 expres-
sion. Cells were treated with 0.1% DMSO (vehicle control), 20 µM taxol, 20 µM
nocodazole, or 20 µM blebbistatin for 60 min before imaging. All blebbistatin
experiments were performed using mApple-EB3 to avoid photo-inactivation
of blebbistatin and phototoxicity from GFP (Sakamoto et al., 2005). For
immunolabeling experiments, cells were fixed 90 min after drug treatment.
2D and 3D cell culture
PA gels were cross-linked to 22 × 22 mm No. 1.5 coverslips (Corning) that
had been preactivated. Preactivation involved first incubating coverslips
Figure 7. MT growth speed is directly cor-
related with branch frequency and inversely cor-
related with branch length. (A) Relationships
between branch frequency (left; fold increase)
or branch length (right; fold increase) and MT
growth speed. r, Pearson correlation coeffi-
cient. (B, left) Outlines of a HUVEC expressing
GFP-EB3 migrating in a 0.7 kPa 3D sandwich
gel at 5 min intervals, color-coded by time as
shown on the far left. (B, right) Color-coded
MT growth track subpopulation overlays from
30-min time-lapse movies of GFP-EB3 (frame
rate = 2 s) from the boxed region of selected
time points during branching morphogenesis.
Arrows, elongating cell branches; arrow-
heads, retracting cell branch. A color key for
MT growth dynamics classifications is shown
below. Bar, 10 µm. (C) Summary table depict-
ing data trends (red triangles/squares) for cell
branching frequency (fold change) compared
with myosin II activity and MT assembly dy-
namics (mean values) on substrates of varying
compliance in 2D and 3D ECMs. (*, Straight
et al., 2003; **, Fischer et al., 2009).
JCB • VOLUME 192 • NUMBER 2 • 2011 332
(1.6 mg/ml; BD). The covalently linked collagen was then polymerized for
4 h at 37°C and rinsed overnight in PBS, and the coverslip with bound col-
lagen was assembled into a Rose chamber. HUVECs expressing fluorescent
EB3 were cultured on the collagen and allowed to adhere (60 min) before
being overlaid with an additional layer of 1.6 mg/ml collagen, which was
subsequently polymerized as described. For live-cell imaging, chambers
were filled with imaging media and sealed with a second coverslip.
2D compliant collagen-coupled PA substrates were assembled as
described for 3D, except that the sulfo-SANPAH activated, PA-coated
coverslips were covalently cross-linked to collagen at a concentration
lower than required for polymerization (90 µg/ml for 8 h at 4°C). After
overnight rinsing, cells were cultured on the collagen and prepared for
imaging as above.
Live-cell imaging of fluorescent EB3. GFP- or mApple-EB3 was imaged on a
spinning disk confocal microscope using a 60× 1.2 NA water immersion
objective lens on an epifluorescence microscope (TE2000; Nikon)
equipped with Perfect Focus System, an electronic shutter (Smart shutter;
Sutter Instrument Co.) for transmitted illumination, a linear-encoded x, y, z
robotic stage with a piezo-driven z-axis top plate (ASI Technologies, Inc.)
and a spinning disk confocal scan head (CSU-X; Yokogawa) equipped
with a multi-bandpass dichromatic mirror (Semrock) and bandpass filters
(Chroma Technology Corp.) in an electronic filter wheel for selection of
GFP or Texas red emission. 561 and 488 nm laser illumination was pro-
vided by a custom-built laser combiner module (modification of LMM-3;
Spectral Applied Research). This contained 500-mW solid-state lasers
(488 nm [Coherent] and 561 nm [MPB Communications]) that were shuttered
with electronic shutters and attenuated and/or directed to a fiber-coupled
output port with an acoustic-optic tunable filter (Neos Technologies), and
directed to the confocal scan head via a single-mode optical fiber (Oz
Optics). Movies of EB3 dynamics were acquired using a cooled charge-
coupled device (CoolSNAP HQ2; Photometrics) operated in the 14-bit
mode for 2 min at 2-s image intervals using a 300-ms exposure time.
Microscope system automation was controlled with MetaMorph software
(MDS Analytical Technologies). For long-term imaging of fluorescent EB3
dynamics during cell branch formation and elongation, 1-min time-lapse
image series (2-s intervals) were captured every 5 min for 30–60 min.
Immunofluorescence. Fixation and processing of samples for immuno-
fluorescence labeling was performed as described previously (Fischer
et al., 2009), with the following modifications. Cells in 3D sandwich gels
were fixed and immunolabeled without disruption of the collagen gel in
3% paraformaldehyde in CB buffer (10 mM MES, pH 6.1, 138 mM KCl,
3 mM MgCl2, and 2 mM EGTA) with 0.1% Triton X-100 for 30 min at
room temperature, then rinsed in CB with 0.2% Triton X-100 for 30 min.
After fixation, cells in PA-collagen gels were blocked with 4% BSA in CB
(3 h, 37°C) and incubated in primary rat anti–-tubulin antibody (overnight
at 4°C, 1:1,000 dilution; AbD Serotec), then rinsed in CB three times for
15 min each before secondary antibodies Cy3-donkey anti–Rat (1:1,000
dilution; Jackson ImmunoResearch Laboratories, Inc.) and Alexa Fluor
488–phalloidin (1:200 dilution; Invitrogen) were applied simultaneously
(2 h, 37°C). After extensive rinsing in CB, PA-collagen gels containing the
cells were imaged directly in the Rose chambers to prevent disruption of
the sandwich gels.
Cell migration assay. Cell migration assays were performed on
22-mm round No. 1.5 coverslips prepared with PA-collagen substrates
as described for 2D and 3D culture, except they were assembled and
mounted in a Gupton chamber, a custom-built multiposition stage insert
that allowed us to image up to 16 coverslips per experiment. Phase-contrast
images were acquired at 15-min intervals for 15 h on the microscope
described above using a 20× 0.45 NA phase objective and an 0.52 NA
LWD condenser using MetaMorph’s Multi-dimensional Acquisition (MDA)
Quantification of cell branching and migration. For analysis of cell
branching, the “trace region” tool in MetaMorph was used to obtain
cell outlines and cell areas. Branches were defined as protrusions that
extended from the cell >10 µm in length. We defined the “branch origin”
by locating the position on each side of the branch where the membrane
displayed the greatest curvature and then connected those two points with
a straight line. The “trace region” tool was then used to trace the outlines
of the branches for each cell to calculate branch area and branch number.
Branch length was the distance from the branch origin to the most distal
point of the branch tip. Cell migration was quantified by hand-tracking
the nucleolus in successive images from a time-lapse phase-contrast image
series using the “track points” application in MetaMorph to determine
instantaneous velocity and distance to origin. Statistical analysis was
performed using the Analyze-It plug-in (Analyze-It Software Ltd.) for Excel
(Microsoft), and branching and migration data were compared using a
Bonferroni-corrected, one-way analysis of variance (ANOVA) test, with
>95% confidence as the threshold for statistical significance.
MT dynamics analysis
MT dynamics were analyzed from EB3 movies using plusTipTracker (Matov
et al., 2010), a Matlab-based, open-source software package that com-
bines automated detection, tracking, analysis, and visualization tools for
movies of fluorescently labeled MT plus end binding proteins (+TIPs). The
+TIP comet detection algorithm relies on a watershed-based approach to
estimate locally optimal thresholds. The track reconstruction algorithm uses
the spatially and temporally globally optimized tracking framework de-
scribed in Jaqaman et al. (2008), with cost functions modified to reflect MT
track geometry. In brief, tracking occurs in two steps: frame-to-frame linking
of comets into growth sub-tracks, and the linking of collinear, sequential
growth sub-tracks into compound tracks. The cost of joining two candidate
growth sub-tracks into a compound track is calculated from three spatial
parameters and one temporal parameter. After calculating the cost of link-
ing all pairs of candidate growth tracks, the links are chosen by minimizing
the global cost, which is achieved by solving the Linear Assignment Prob-
lem (Jaqaman et al., 2008).
Determination of image quality. Detection, tracking, and postprocess-
ing analysis were performed on the first 30 frames only for each movie, as
photo-bleaching was a limiting feature in some conditions and standardiz-
ing movie length was required for the MT tracking comparison. The quality
of the movies was assessed by examining comet detection performance;
movies were discarded from further analysis if EB3 expression was too
high and led to too many false positives, or if focus drift or photobleaching
led to a high standard deviation in mean comet number per frame over the
course of the movie.
Tracking parameters. Tracking control parameters were optimized
based on a parameter sweep using the plusTipParamSweepGUI tool of
plusTipTracker and verified by visual inspection of track overlays on mov-
ies. The same parameter set was used for all movies in the dataset: maxi-
mum gap length, 12 frames; minimum track length, 3 frames; search radius
range, 5–10 pixels; maximum forward angle, 25°; maximum backward
angle, 8°; maximum shrinkage factor, 1.0; fluctuation radius, 2 pixels. For
this study, only growth excursions were of interest, so MT shrinkage or
pause events were not analyzed. However, sub-track linking was still per-
formed to correct for the many occurrences when comets cross over one
another or disappear momentarily from the field of view by focal drift,
which breaks the trajectories prematurely.
Region of interest (ROI) selection. Binary masks of whole cells, indi-
vidual branches, and cell peripheries were generated in two steps using
plusTipTracker’s sub-ROI selection tool. First, whole cell and branch masks
were manually selected based on the whole cell outlines and branch defi-
nitions were produced in MetaMorph as described above. In the second
step, cell periphery masks were automatically generated from whole cell
masks by including all pixels within 10 µm of the cell edge but not includ-
ing pixels defined within branches. The subtraction of branch pixels was
accomplished by choosing the all-branch mask generated in the first step
as an exclusion mask in the second. Regional areas were calculated by
summing the area of the mask and converting to square micrometers. The
set of all MT growth excursions that spent ≥6s (three frames) within an ROI
(branch or periphery) were extracted and stored within a subproject of the
Data grouping. The plusTipPickGroups function of plusTipTracker was
used to create groups of data defined by experimental condition (glass [2D
only], 55 kPa [2D or 3D], 8.7 kPa [2D or 3D], or 0.7 kPa [2D or 3D]; drug
or vehicle) and cellular region (whole cell, branch, or peripheral cell body).
There were thus 42 groups to be compared, 14 each from the three re-
gions. Groups of whole cells and peripheries ranged from 8 to 17 projects,
whereas groups of branches ranged from 2 to 57 projects.
MT growth sub-track subpopulation analysis. Tracks from within ROIs
from all movies in the dataset were pooled using the plusTipPoolGroup-
Data function to find the mean growth speed and mean growth lifetime.
These values were used to split data for quadrant plot analysis. Quadrant
plot analysis was performed as separate batch processes for the three re-
gions, thus defining for each of the groups the total number of tracks in
each of four subpopulations: slow and short-lived, slow and long-lived, fast
and short-lived, and fast and long-lived. The relative proportions of these
four subpopulations were used to generate percentage bar graphs for com-
parison. Statistical comparison of MT growth speeds and growth lifetimes
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Online supplemental material
Fig. S1 shows whole cell and regional MT track density in either branches
or peripheral cell bodies as detected by plusTipTracker software. Fig. S2
shows total track numbers for each category in every experimental con-
dition, and the total correlated area associated with each experimental
condition. Fig. S3 is the summary of all MT growth excursion class propor-
tions for all MT populations and experimental conditions analyzed. Fig. S4
shows correlation analyses for MT growth lifetimes and EC branch fre-
quency or branch length under all conditions analyzed. Table S1 lists all
MT growth speeds and growth lifetimes under all experimental conditions,
with associated means and standard errors. Video 1 shows HUVEC mi-
gration on glass with DMSO, Taxol, or nocodazole treatments. Video 2
shows HUVEC migration on 8.7 kPa 2D substrates with DMSO, Taxol, or
nocodazole treatments. Video 3 shows HUVEC migration on 0.7 kPa 2D
substrates with DMSO, Taxol, or nocodazole treatments. Videos 4 and 5
show HUVEC migration on 8.7 kPa and 0.7 kPa 3D sandwich gels with
DMSO, Taxol, or nocodazole treatments. Video 6 compares GFP-EB3 dy-
namics in HUVECs on 8.7 kPa 2D and 8.7 kPa 3D sandwich gel substrates.
Video 7 compares GFP-EB3 dynamics in HUVECs on 0.7 kPa 3D sandwich
gels with EB3-GFP dynamics in HUVECs treated with blebbistatin on 8.7 kPa
3D sandwich gels. Video 8 shows long-term imaging of EC branching morpho-
genesis and GFP-EB3 dynamics. Online supplemental material is available
We thank Mike Davidson for fluorescent EB3, William Shin for maintaining the
microscopes, Dorothy Honemond for administrative assistance, and members
of the Waterman laboratory and Orna Cohen-Fix (National Institute of Diabe-
tes and Digestive and Kidney Diseases) for helpful discussions.
This work was initiated as a project in the Physiology Course at the
Marine Biological Laboratory in Woods Hole, MA, and we thank student
Julie Janvore (Institute Curie, Paris, France) for her work at early stages of
the study. C.M. Waterman, R.S. Fischer, and K.A. Myers are supported
by the National Heart, Lung and Blood Institute, and G. Danuser and K.T.
Applegate are supported by National Institute of General Medical Sciences
Submitted: 1 June 2010
Accepted: 23 December 2010
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